The unpredictability and expense of conventional drug discovery has led to the exploration of several drug discovery approaches that promise more systematic and/or rapid identification candidate compounds for testing in biological assays and disease models. Examples of such approaches include selection of small peptides from a synthetic or recombinant peptide libraries, e.g. Pirrung et al, U.S. Pat. No. 5,143,854; Geysen et al, J. Immunol. Meth., 102: 259-274 (1987); Lam et al, Nature, 354: 82-84 (1991); Scott et al, Science, 249: 386-390 (1990); the construction and selection of human or humanized antibodies from recombinant antibody libraries, e.g. Riechmann et al, Nature, 332: 323-327 (1988); Winter and Milstein, Nature, 349: 293-299 (1991); selection of aptamers or ribozymes from random sequence polynucleotide libraries, e.g. Ellington and Szostak, Nature, 346: 818-822 (1990); Blackwell et al, Science, 250: 1104-1110 (1990); Tuerk et al, Science, 249: 505-510 (1990); Joyce, Gene, 82: 83-87 (1989); Cech et al, U.S. Pat. No. 4,987,071; Haseloff et al, Nature, 334: 585-591 (1988); and the use of antisense oligonucleotides, e.g. Uhlmann and Peyman, Chemical Reviews, 90: 543-584 (1990); Goodchild, Bioconjugate Chemistry, 1: 165-187 (1990); Helene et al, Biochim. Biophys. Acta, 1049: 99-125 (1990); Cohen, Ed., Oligonucleotides: Antisense Inhibitors of Gene Expression (Macmillan Press, New York, 1989); Crooke, Ann. Rev. Pharmacol. Toxicol., 32: 329-376 (1992); McManaway et al, Lancet, Vol. 335, pgs. 808-811 (1990); Bayever et al, Antisense Research and Development, 2: 109-110 (1992); Manson et al, Lymphokine Research, Vol. 9, pgs. 35-42 (1990); Lisziewicz et al, Proc. Natl. Acad. Sci., 90: 3860-3864 (1993); Miller, Biotechnology, Vol. 9, pgs. 358-362 (1991); Chiang et al, J. Biol. Chem., Vol. 266, pgs. 18162-18171 (1991); Calabretta, Cancer Research, Vol. 51, pgs. 4505-4510 (1991); and the like.
Of the cited examples, the antisense approach presents a compelling advantage of not requiring one or more initial screening steps to identify candidate compounds capable of binding to a predetermined target. Specific binding is achieved by providing an oligonucleotide or an analog thereof capable of forming a stable duplex or triplex with a target nucleotide sequence based on Watson-Crick or Hoogsteen binding, respectively. Thus, as soon as the sequence of a target polynucleotide is determined, the structure of candidate antisense compounds is also determined. The specifically bound antisense compound then either renders the respective targets more susceptible to enzymatic degradation, blocks translation or processing, or otherwise blocks or inhibits its expression or function.
Another advantage of the antisense approach has been the development of reliable and convenient methods for solid phase synthesis of polynucleotides and analogs thereof, e.g. Caruthers, Science, Vol. 230, pgs 281-285 (1985); Beaucage et al, Tetrahedron, 48: 2223-2311 (1992); and Eckstein, ed., Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991). In particular, the availability of synthetic oligonucleotides and a variety of nuclease-resistant analogs, e.g. phosphorothioates, methylphosphonates, and the like, has further encouraged investigation of antisense compounds for treating a variety of conditions associated with the inappropriate expression of indigenous and/or exogenous genes, such as described in the references cited above.
Notwithstanding the many hurdles that have been overcome in the course of developing antisense compounds, several significant uncertainties still stand in the way of their widespread application as drugs. One such uncertainty concerns whether a sufficient concentration of antisense compound can be delivered to its target polynucleotide so that translation or transcription can be effectively shut down. This problem has led to many proposals for enhancing oligonucleotide drug delivery, e.g. Letsinger, U.S. Pat. No. 4,958,013; Latham et al, International application PCT/US91/02224; MacKellar et al, Nucleic Acids Research, 20: 3411-3417 (1992); Wagner et al, Proc. Natl. Acad. Sci., 87: 3410-3414 (1990); Citro et al, Proc. Natl. Acad. Sci., 89: 703 1-7035 (1992); Rosenberg et al, International application PCT/US92/05305. One aspect of this problem is the observation that longer oligonucleotides appear to be more difficult to deliver to cellular interiors than shorter oligonucleotides, e.g. Loke et al,; Proc. Natl. Acad. Sci., 86: 3474-3478 (1989); and Maher III et al, Nucleic Acids Research, 16: 3341-3357 (1988). On the other hand, it has also been observed that shorter antisense compounds appear to be less effective in inhibiting expression than longer antisense compounds, apparently due to the lack of stability of the shorter duplexes under physiological conditions. Thus, with current approaches, a serious trade-off exists in selecting the "best" size of antisense compound.
Another uncertainty concerns the degree of specificity of antisense oligonucleotides under physiological conditions. Antisense oligonucleotides could be non-specific in at least two senses: (i) duplex or triplex formation may lack specificity, e.g. non-perfectly matched duplexes may form--leading to the unwanted inhibition of non-target polynucleotides, and (ii) the moieties not directly involved in base pairing, e.g. the backbone or other appending groups, may interact non-specifically with other cellular components leading to undesired side effects, e.g. Woolf et al, Proc. Natl. Acad. Sci., 89: 7305-7309 (1992); Matsukura et al, Proc. Natl. Acad. Sci., 84:7706-7710 (1987); and the like. In regard to first type of nonspecificity, it has been observed that duplexes involving longer oligonucleotides tend to be more tolerant of mismatches--and hence, less specific--than duplexes involving shorter oligonucleotides, e.g. Young et al, Nucleic Acids Research, 19: 2463-2470 (1991). In regard to the second type of nonspecificity, such activity may not be surprising in view of the large body of work on the use of polyanions, in particular homopolymeric polynucleotides, as anti-viral compounds, e.g. Levy, Chapter 7, in Stringfellow, editor, Interferon and Interferon Inducers (Marcel Dekker, New York, 1980). Interestingly, increased activity--and with some polyanions increased toxicity--has been observed with increased polymer size.
Several of the problems summarized above could be obviated by the availability of a means for remotely assembling antisense compounds in situ from shorter fragments which separately could be delivered to a target polynucleotide and which would have reduced non-specific binding because of the unfavorable binding kinetics of short oligonucleotides for partially complementary sequences under physiological conditions.